Inhibitors of protein farnesyl transferase

ABSTRACT

The present invention provides compounds having the structure represented by Formula I:                    
     and methods of treating cancer, restenosis, atherosclerosis, or psoriasis or preventing restenosis, and a pharmaceutical composition comprising a compound of Formula I and a pharmaceutically acceptable carrier.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/331,876 filed Jun. 28, 1999, now allowed, which is a §371 ofPCT/US98/06482 filed Apr. 2, 1998, and claims priority from U.S.Provisional Application No. 60/043,492 filed Apr. 11, 1997.

FIELD OF THE INVENTION

The present invention relates to compounds that can be used in themedicinal field to treat, prophylactically or otherwise, uncontrolled orabnormal proliferation of human tissues. Specifically, the presentinvention relates to compounds that inhibit the farnesyl transferaseenzyme, which has been determined to activate ras proteins that in turnactivate cellular division and are implicated in cancer, restenosis,atherosclerosis, and psoriasis.

BACKGROUND OF THE INVENTION

Ras protein (or p21) has been examined extensively because mutant formsare found in 20% of most types of human cancer and greater than 50% ofcolon and pancreatic carcinomas (Gibbs J. B., Cell, 1991;65:1,Cartwright T., et al., Chimica. Oggi., 1992;10:26). These mutant rasproteins are deficient in the capability for feedback regulation that ispresent in native ras, and this deficiency is associated with theironcogenic action since the ability to stimulate normal cell divisioncannot be controlled by the normal endogenous regulatory cofactors. Therecent discovery that the transforming activity of mutant ras iscritically dependent on posttranslational modifications (Gibbs J., etal., Microbiol. Rev., 1989;53:171) has unveiled an important aspect ofras function and identified novel prospects for cancer therapy.

In addition to cancer, there are other conditions of uncontrolledcellular proliferation that may be related to excessive expressionand/or function of native ras proteins. Postsurgical vascular restenosisis such a condition. The use of various surgical revascularizationtechniques such as saphenous vein bypass grafting, endarterectomy, andtransluminal coronary angioplasty are often accompanied by complicationsdue to uncontrolled growth of neointimal tissue, known as restenosis.The biochemical causes of restenosis are poorly understood and numerousgrowth factors and protooncogenes have been implicated (Naftilan A. J.,et al., Hypertension, 1989;13:706 and J. Clin. Invest., 83:1419; GibbonsG. H., et al., Hypertension, 1989;14:358; Satoh T., et al., Molec. Cell.Biol., 1993;13:3706). The fact that ras proteins are known to beinvolved in cell division processes makes them a candidate forintervention in many situations where cells are dividing uncontrollably.In direct analogy to the inhibition of mutant ras related cancer,blockade of ras dependent processes has the potential to reduce oreliminate the inappropriate tissue proliferation associated withrestenosis, particularly in those instances where normal ras expressionand/or function is exaggerated by growth stimulatory factors.

Ras functioning is dependent upon the modification of the proteins inorder to associate with the inner face of plasma membranes. Unlike othermembrane-associated proteins, ras proteins lack conventionaltransmembrane or hydrophobic sequences and are initially synthesized ina cytosol soluble form. Ras protein membrane association is triggered bya series of posttranslational processing steps that are signaled by acarboxyl terminal amino acid consensus sequence that is recognized byprotein farnesyl transferase (PFT). This consensus sequence consists ofa cysteine residue located four amino acids from the carboxyl terminus,followed by two lipophilic amino acids and the C-terminal residue. Thesulfhydryl group of the cysteine residue is alkylated byfarnesylpyrophosphate in a reaction that is catalyzed by proteinfarnesyl transferase. Following prenylation, the C-terminal three aminoacids are cleaved by an endoprotease and the newly exposedalpha-carboxyl group of the prenylated cysteine is methylated by amethyl transferase. The enzymatic processing of ras proteins that beginswith farnesylation enables the protein to associate with the cellmembrane. Mutational analysis of oncogenic ras proteins indicates thatthese posttranslational modifications are essential for transformingactivity. Replacement of the consensus sequence cysteine residue withother amino acids gives a ras protein that is no longer farnesylated,fails to migrate to the cell membrane and lacks the ability to stimulatecell proliferation (Hancock J. F., et al., Cell, 1989;57:1617, SchaferW. R., et al., Science, 1989;245:379, Casey P. J., Proc. Natl. Acad.Sci. USA, 1989;86:8323).

Recently, protein farnesyl transferases (PFTs), also referred to asfarnesyl proteintransferases (FPTs) have been identified, and a specificPFT from rat brain was purified to homogeneity (Reiss Y., et al.,Biochem. Soc. Trans., 1992;20:487-88). The enzyme was characterized as aheterodimer composed of one alpha-subunit (49 kDa) and one beta-subunit(46 kDa), both of which are required for catalytic activity. High levelexpression of mammalian PFT in a baculovirus system and purification ofthe recombinant enzyme in active form has also been accomplished (Chen,W. J., et al., J. Biol. Chem., 1993;268:9675).

In light of the foregoing, the discovery that the function of oncogenicras proteins is critically dependent on their posttranslationalprocessing provides a means of cancer chemotherapy through inhibition ofthe processing enzymes. The identification and isolation of a proteinfarnesyl transferase that catalyzes the addition of a farnesyl group toras proteins provides a promising target for such intervention. Rasfarnesyl transferase inhibitors have been shown to have anticanceractivity in several recent articles.

Ras inhibitor agents act by inhibiting farnesyl transferase, the enzymethat anchors the protein product of the ras gene to the cell membrane.The role of the ras mutation in transducing growth signals within cancercells relies on the protein being in the cell membrane, so with farnesyltransferase inhibited, the ras protein will stay in the cytosol and beunable to transmit growth signals: these facts are well-known in theliterature.

A peptidomimetic inhibitor of farnesyl transferase B956 and its methylester B1086 at 100 mg/kg have been shown to inhibit tumor growth by EJ-1human bladder carcinoma, HT1080 human fibrosarcoma, and human coloncarcinoma xenografts in nude mice (Nagasu, T., et al., Cancer Res.,1995;55:5310-5314). Furthermore, inhibition of tumor growth by B956 hasbeen shown to correlate with inhibition of ras posttranslationalprocessing in the tumor. Other ras farnesyl transferase inhibitors havebeen shown to specifically prevent ras processing and membranelocalization and are effective in reversing the transformed phenotype ofmutant ras containing cells (Sepp-Lorenzino L., et al., Cancer Res.,1995;55:5302-5309).

In another report (Sun J., et al., Cancer Res., 1995;55:4243-4247), aras farnesyl transferase inhibitor FT1276 has been shown to selectivelyblock tumor growth in nude mice of a human lung carcinoma with K-rasmutation and p53 deletion. In yet another report, daily administrationof a ras farnesyl transferase inhibitor L744,832 caused tumor regressionof mammary and salivary carcinomas in ras transgenic mice (Kohl et al.,Nature Med., 1995; 1(8):792-748). Thus, ras farnesyl transferaseinhibitors have benefit in certain forms of cancer, particularly thosedependent on oncogenic ras for their growth. However, it is well-knownthat human cancer is often manifested when several mutations inimportant genes occur, one or more of which may be responsible forcontrolling growth and metastases. A single mutation may not be enoughto sustain growth and only after two or three mutations occur, tumorscan develop and grow. It is therefore difficult to determine which ofthese mutations may be primarily driving the growth in a particular typeof cancer. Thus, ras farnesyl transferase inhibitors can havetherapeutic utility in tumors not solely dependent on oncogenic forms ofras for their growth. For example, it has been shown that various rasFT-inhibitors have antiproliferative effects in vivo against tumor lineswith either wild-type or mutant ras (Sepp-Lorenzino, supra). Inaddition, there are several ras-related proteins that are prenylated.Proteins such as R-Ras2/TC21 are ras-related proteins that areprenylated in vivo by both farnesyl transferase and geranylgeranyltransferase I (Carboni, et al., Oncogene, 1995;10:1905-1913). Therefore,ras farnesyl transferase inhibitors could also block the prenylation ofthe above proteins and therefore would then be useful in inhibiting thegrowth of tumors driven by other oncogenes.

With regard to restenosis and vascular proliferative diseases, it hasbeen shown that inhibition of cellular ras prevents smooth muscleproliferation after vascular injury in vivo (Indolfi C., et al., NatureMed., 1995;1(6):541-545). This report definitively supports a role forfarnesyl transferase inhibitors in this disease, showing inhibition ofaccumulation and proliferation of vascular smooth muscle.

SUMMARY OF THE INVENTION

Provided by the present invention are compounds having the Formula I

wherein

R^(a), R^(b), and R^(c) are each independently C₁-C₆ alkyl or hydrogen;

R^(d), R^(e), R^(f), and R^(g) are each independently C₁-C₆ alkyl,hydrogen, or phenyl;

R⁴ is aryl, substituted aryl, or C₁-C₆ alkyl; and

each n is independently 0 to 5, m is 2 to 4, and the pharmaceuticallyacceptable salts, and prodrugs thereof.

In a preferred embodiment of Formula I, Y is —O—.

In another preferred embodiment of Formula I,

In another preferred embodiment of Formula I, R^(a) is hydrogen, R^(b)is methyl, and R^(c) is hydrogen.

In another preferred embodiment of Formula I, R³ is

In another preferred embodiment of Formula I, R³ is

In another preferred embodiment of Formula I, R² is —CH₂CH₂-phenyl or—CH₂CH₂-substituted phenyl.

In another preferred embodiment of Formula I,

In another preferred embodiment of Formula I,

In another preferred embodiment of Formula I, R³ is —(CH₂)_(n)—C₁-C₆alkyl.

In another preferred embodiment of Formula I, R³ is

In a more preferred embodiment, the present invention provides thefollowing compounds:

[S-(R*,R*)]-[1-{[2-(4-Benzyloxy-phenyl)-1-phenethylcarbamoyl-ethyl]-methyl-carbamoyl}-2-(1H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester;

[S-(R*,R*)]-[1-{[2-(4-Benzyloxy-phenyl)-1-(2-methyl-2-phenyl-propylcarbamoyl)-ethyl]-methyl-carbamoyl}-2-1H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester;

[S-(R*,R*)]-[2-1H-Imidazol-4-yl)-1-(methyl-{1-(2-methyl-2-phenyl-propylcarbamoyl)-2-[4-(pyridin-2-ylmethoxy)-phenyl]-ethyl}-carbamoyl)-ethyl]-carbamicacid benzyl ester;

[S-(R*,R*)]-2-(3-Benzyl-3-methyl-ureido)-N-[2-(4-benzyloxy-phenyl)-1-(2-methyl-2-phenyl-propyl-carbamoyl)-ethyl]-3-(1H-imidazol-4-yl)-N-methyl-propionamide;

[S-(R*,R*)]-[1-{[2-(4-Benzyloxy-phenyl)-1-[(1-phenyl-cyclobutylmethyl)-carbamoyl]-ethyl}-methyl-carbamoyl)-2-(3H-imidazol-4-yl)-ethyl]-carbanicacid benzyl ester;

[S-(R*,R*)]-[2-(3H-Imidazol-4-yl)-1-{methyl-[1-(2-methyl-2-phenyl-propylcarbamoyl)-2-phenyl-ethyl]-carbarmoyl}ethyl-carbamicacid benzyl ester; and

[S-(R*,R*)]-[2-(3H-Imidazol-4-yl)-1-{methyl-[3-methyl-1-(2-methyl-2-phenyl-propylcarbamoyl)-butyl]-carbamoyl}-ethyl-carbamicacid benzyl ester.

In another more preferred embodiment, the present invention provides thefollowing compounds:

[S-(R*,R*)]-[1-({2-(4-Benzyloxy-phenyl)-1-[2-(2-fluoro-phenyl)-ethyl-carbamoyl]-ethyl}-methyl-carbamoyl)-2-(3H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester;

[S-(R*,R*)]-[1-{[2-(4-Benzyloxy-phenyl)-1-(2-pyridin-2-yl-ethyl-carbamoyl]-ethyl]-methyl-carbamoyl}-2-(3H-imidazol-4-yl)-ethyl]-carbamic acid benzyl ester;

[S-(R*,R*)]-[1-{[2-(4-Benzyloxy-phenyl)-1-(2,2-diphenyl-ethylcarbamoyl)-ethyl]-methyl-carbamoyl}-2-(3H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester;

[S-(R*,R*)]-[1-{[2-(4-Benzyloxy-phenyl)-1-(2-phenyl-propylcarbamoyl)-ethyl]-methyl-carbamoyl}-2-(3H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester;

[S-(R*,R*)]-(2-(3H-Imidazol-4-yl)-1-{methyl-[3-methyl-1-(2-methyl-2-phenyl-propylcarbamoyl)-butyl]-carbamoyl}-ethyl)-carbamicacid benzyl ester;

[S-(R*,R*)]-[1-{[2-(4-Benzyloxy-phenyl)-1-(1-methyl-2-phenyl-ethylcarbamoyl)-ethyl]-methyl-carbamoyl}-2-(3H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester;

[S-(R*,R*)]-[1-({2-(4-Benzyloxy-phenyl)-1-[(1-phenyl-cyclopropyl-methyl)-carbamoyl]-ethyl}-methyl-carbamoyl)-2-(3H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester;

[S-(R*,R*)]-[1-{[2-(4-Chloro-phenyl)-1-(2-methyl-2-phenyl-propylcarbamoyl)-ethyl]-methyl-carbamoyl}-2-(3H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester;

[S-(R*,R*)]-2-(3-Benzyl-ureido)-3-(3H-imidazol-4-yl)-N-methyl-N-{1-(2-methyl-propyl-carbamoyl)-2-[4-(pyridin-4-ylmethoxy)-phenyl]-ethyl}-propionamide;

[S-(R*,R*)]-[1-{[2-(4-Benzyloxy-phenyl)-1-(1-methyl-2-phenyl-ethylcarbamoyl)-ethyl]-methyl-carbamoyl}-2-(3H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester;

[S-(R*,R*)]-(2-(3H-Imidazol-4-yl)-1-{methyl-[1-(2-methyl-2-phenyl-propylcarbamoyl)-2-p-tolyl-ethyl]-carbamoyl}-ethyl)-carbamicacid benzyl ester;

[S-(R*,R*)]-(2-(3H-Imadozol-4-yl)-1-{[2-(4-methoxy-phenyl)-1-(2-methyl-2-phenyl-propylcarbamoyl)-ethyl]-methyl-carbamoyl}-ethyl)-carbamicacid benzyl ester;

[S-(R*,R*)]-2-(3-Benzyl-ureido)-3-(3H-imidazol-4-yl)-N-methyl-N-[1-(2-methyl-2-phenyl-propylcarbamoyl)-2-phenyl-ethyl]-propionamide;and

[S-(R*,R*)]-[1-[(2-(4-Benzyloxy-phenyl)-1-{[1-(2-fluoro-phenyl)-cyclopropylmethyl]-carbamoyl}ethyl)-methyl-carbamoyl]-2-(3H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester.

Also provided is a method of treating cancer, the method comprisingadministering to a patient having cancer a therapeutically effectiveamount of a compound of Formula I.

Also provided is a method of treating or preventing restenosis, themethod comprising administering to a patient having restenosis or atrisk of having restenosis a therapeutically effective amount of acompound of Formula I.

Also provided is a method of treating atherosclerosis, the methodcomprising administering to a patient having atherosclerosis atherapeutically effective amount of a compound of Formula I.

Also provided is a method of treating psoriasis, the method comprisingadministering to a patient having psoriasis a therapeutically effectiveamount of a compound of Formula I.

Also provided is a pharmaceutical composition that comprises a compoundof Formula I.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds having the Formula I.

wherein

R^(a) R^(b), and R^(c) are each independently C₁-C₆ alkyl or hydrogen;

R^(d), R^(e), R^(f), and R^(g) are each independently C₁-C₆ alkyl,hydrogen, or phenyl;

R⁴ is aryl, substituted aryl, or C₁-C₆ alkyl; and each n isindependently 0 to 5, m is 2 to 4, and the pharmaceutically acceptablesalts, and prodrugs thereof.

The term “alkyl” means a straight or branched hydrocarbon having from 1to 6 carbon atoms and includes, for example, methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl,and the like.

The alkyl groups of the present invention include substituted alkylgroups. Examples of suitable substituents include, but are not limitedto, halogen, —OC₁-C₆ alkyl, —SC₁-C₆ alkyl, _(CF) ₃, —NO₂, —CN, phenyl,—OH, —SH, —NH₂, —NHC₁-C₆ alkyl, or —N(C₁-C₆ alkyl)₂.

The term “aryl” means an aromatic ring which is a phenyl, 5-fluorenyl,1-naphthyl, or 2-naphthyl group, unsubstituted or substituted by 1 to 3substituents selected from alkyl, phenyl, O-phenyl, O-alkyl and S-alkyl,OH, SH, F, Cl, Br, I, CF₃, NO₂, NH₂, NHCH₃, N(CH₃)₂, NHCO-alkyl,(CH₂)_(m)CO₂H, NHC₁-C₆ alkyl, N(C₁-C₆ alkyl)₂, (CH₂)_(m)CO₂-alkyl,(CH₂)_(m)SO₃H, (CH₂)_(m)PO₃H₂, (CH₂)_(m)PO₃(alkyl)₂, (CH₂)_(m)SO₂NH₂,and (CH₂)_(m)SO₂NH-alkyl wherein alkyl is defined as above and m=0, 1,2, or 3.

The term “heteroaryl” means a heteroaromatic ring which is a 2- or3-thienyl, 2- or 3-furanyl, 2- or 3-pyrrolyl, 2-, 3-, or 4-pyridyl,imidazolyl, 2-, 3-, 4-, 5-, 6-, or 7-indoxyl group, unsubstituted orsubstituted by 1 or 2 substituents from the group of substituentsdescribed above for aryl.

The symbol “—” means a bond.

The term “patient” means all animals including humans. Examples ofpatients include humans, cows, dogs, cats, goats, sheep, and pigs.

A “therapeutically effective amount” is an amount of a compound of thepresent invention that when administered to a patient ameliorates asymptom of restenosis, cancer, atherosclerosis, or psoriasis, orprevents restenosis. A therapeutically effective amount of a compound ofthe present invention can be easily determined by one skilled in the artby administering a quantity of a compound to a patient and observing theresult. In addition, those skilled in the art are familiar withidentifying patients having cancer, restenosis, atherosclerosis, orpsoriasis, or who are at risk of having restenosis.

The term “cancer” includes, but is not limited to, the followingcancers:

breast;

ovary;

cervix;

prostate;

testis;

esophagus;

glioblastoma;

neuroblastoma;

stomach;

skin, keratoacanthoma;

lung, epidermoid carcinoma, large cell carcinoma, adenocarcinoma;

bone;

colon, adenocarcinoma, adenoma;

pancreas, adenocarcinoma;

thyroid, follicular carcinoma, undifferentiated carcinoma, papillarycarcinoma;

seminoma;

melanoma;

sarcoma;

bladder carcinoma;

uterine;

liver carcinoma and biliary passages;

kidney carcinoma;

myeloid disorders;

lymphoid disorders, Hodgkins, hairy cells; buccal cavity and pharynx(oral), lip, tongue, mouth, pharynx;

small intestine;

colon-rectum, large intestine, rectum;

brain and central nervous system; and

leukemia.

The term “pharmaceutically acceptable salts, and prodrugs” as usedherein refers to those carboxylate salts, amino acid addition salts, andprodrugs of the compounds of the present invention which are, within thescope of sound medical judgment, suitable for use in contact with thetissues of patients without undue toxicity, irritation, allergicresponse, and the like, commensurate with a reasonable benefit/riskratio, and effective for their intended use, as well as the zwitterionicforms, where possible, of the compounds of the invention. The term“salts” refers to the relatively nontoxic, inorganic and organic acidaddition salts of compounds of the present invention. These salts can beprepared in situ during the final isolation and purification of thecompounds or by separately reacting the purified compound in itsfree-base form with a suitable organic or inorganic acid and isolatingthe salt thus formed. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate,oleate, palmitate, stearate, laureate, borate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,naphtholate mesylate, glucoheptonate, lactobionate and laurylsulphonatesalts, and the like. These may include cations based on the alkali andalkaline earth metals, such as sodium, lithium, potassium, calcium,magnesium and the like, as well as nontoxic ammonium, quaternaryammonium, and amine cations including, but not limited to ammonium,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, triethylamine, ethylamine, and the like. (See, forexample, Berge, S. M. et al., “Pharmaceutical Salts,” J. Pharm. Sci.,1977;66:1-19 which is incorporated herein by reference.)

The term “prodrug” refers to compounds that are rapidly transformed invivo to yield the parent compound of the above formulae, for example, byhydrolysis in blood. A thorough discussion is provided in Higuchi, T.and Stella, V., “Pro-drugs as Novel Delivery Systems,” Vol 14 of theA.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design,ed. Edward, B. Roche, American Pharmaceutical Association and PergamonPress, 1987, both of which are hereby incorporated by reference.

The compounds of the present invention can be administered to a patientalone or as part of a composition that contains other components such asexcipients, diluents, and carriers, all of which are well-known in theart. The compositions can be administered to humans and animals eitherorally, rectally, parenterally (intravenously, intramuscularly, orsubcutaneously), intracisternally, intravaginally, intraperitoneally,intravesically, locally (powders, ointments, or drops), or as a buccalor nasal spray.

Compositions suitable for parenteral injection may comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents,solvents, or vehicles include water, ethanol, polyols (propyleneglycol,polyethyleneglycol, glycerol, and the like), Cremophor EL (a derivativeof castor oil and ethylene oxide; purchased from Sigma Chemical Co., St.Louis, Mo.) and suitable mixtures thereof, vegetable oils (such as oliveoil), and injectable organic esters such as ethyl oleate. Properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersions and by the use of surfactants.

These compositions may also contain adjuvants such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, forexample sugars, sodium chloride, and the like. Prolonged absorption ofthe injectable pharmaceutical form can be brought about by the use ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is admixed with at least one inert customary excipient (orcarrier) such as sodium citrate or dicalcium phosphate or (a) fillers orextenders, as for example, starches, lactose, sucrose, glucose,mannitol, and silicic acid; (b) binders, as for example,carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone,sucrose, and acacia; (c) humectants, as for example, glycerol; (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates, andsodium carbonate, (e) solution retarders, as for example paraffin; (f)absorption accelerators, as for example, quaternary ammonium compounds;(g) wetting agents, as for example, cetyl alcohol and glycerolmonostearate; (h) adsorbents, as for example, kaolin and bentonite; and(i) lubricants, as for example, talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, or mixturesthereof. In the case of capsules, tablets, and pills, the dosage formsmay also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethyleneglycols, andthe like.

Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others well-known in the art. They may contain opacifyingagents, and can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions which can be used arepolymeric substances and waxes. The active compounds can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the active compounds, the liquid dosage forms may containinert diluents commonly used in the art, such as water or othersolvents, solubilizing agents and emulsifiers, as for example, ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,dimethylformamide, oils, in particular, cottonseed oil, groundnut oil,corn germ oil, olive oil, castor oil and sesame oil, glycerol,tetrahydrofurfuryl alcohol, Cremophor EL (a derivative of castor oil andethylene oxide; purchased from Sigma Chemical Co., St. Louis, Mo.),polyethyleneglycols and fatty acid esters of sorbitan or mixtures ofthese substances, and the like.

Besides such inert diluents, the composition can also include adjuvants,such as wetting agents, emulsifying and suspending agents, sweetening,flavoring and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspendingagents, as for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, or mixtures of thesesubstances, and the like.

Compositions for rectal administrations are preferably suppositorieswhich can be prepared by mixing the compounds of the present inventionwith suitable nonirritating excipients or carriers such as cocoa butter,polyethyleneglycol, or a suppository wax, which are solid at ordinarytemperatures but liquid at body temperature and therefore, melt in therectum or vaginal cavity and release the active component.

Dosage forms for topical administration of a compound of this inventioninclude ointments, powders, sprays, and inhalants. The active componentis admixed under sterile conditions with a physiologically acceptablecarrier and any preservatives, buffers, or propellants as may berequired. Ophthalmic formulations, eye ointments, powders, and solutionsare also contemplated as being within the scope of this invention.

The compounds of the present invention can be administered to a patientat dosage levels in the range of about 0.1 to about 2,000 mg per day.For a normal human adult having a body weight of about 70 kg, a dosagein the range of about 0.01 to about 100 mg/kg of body weight per day ispreferable. The specific dosage used, however, can vary. For example,the dosage can depend on a numbers of factors including the requirementsof the patient, the severity of the condition being treated, and thepharmacological activity of the compound being used. The determinationof optimum dosages for a particular patient is well known to thoseskilled in the art.

The compounds of the present invention can exist in differentstereoisomeric forms by virtue of the presence of asymmetric centers inthe compounds. It is contemplated that all stereoisomeric forms of thecompounds as well as mixtures thereof, including racemic mixtures, formpart of this invention.

In addition, the compounds of the present invention can exist inunsolvated as well as solvated forms with pharmaceutically acceptablesolvents such as water, ethanol, and the like. In general, the solvatedforms are considered equivalent to the unsolvated forms for the purposesof the present invention.

The examples presented below are intended to illustrate particularembodiments of the invention, and are not intended to limit the scope ofthe specification or the claims in any way.

Schemes 1 through 8 below show generally how compounds of the presentinvention can be synthesized.

Scheme 1 illustrates a general method by which some of these compoundscan be prepared, by illustrating the synthesis of[R,-(R*,S*)]-[1-{[1-(2-benzyloxy-ethylcarbamoyl)-2-(4-benzyloxy-phenyl)-ethyl]-methyl-carbamoyl}-2-(1H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester (Example 1). Coupling of Boc-NMe-Tyr(OBn)OH to2-(phenylmethoxy)-ethylamine hydrochloride was carried out in ethylacetate with dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole(HOBt), as coupling agents, and triethylamine as the base. The resultingproduct was treated with 30% trifluoroacetic acid (TFA) in methylenechloride. H-NMe-Tyr(OBn)-NH—CH₂—CH₂-OBn.TFA was then coupled inmethylene chloride to Cbz-DHis(Trt)-OH, withbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP) as coupling agent, and diisopropylethylamine (DIEA) as the base,followed by treatment with 50% TFA in methylene chloride to give thetitle compound.

2-(Phenylmethoxy)-ethylamine hydrochloride was prepared by reacting2-ethanolamine hydrochloride with sodium, followed by benzyl chloride,in toluene. The product was isolated as the HCl salt.

Scheme 2 illustrates a general method by which some of these compoundscan be prepared, by illustrating the synthesis of[S-(R*,R*)]-[1-{[2-(4-benzyloxy-phenyl)-1-phenethylcarbamoyl-ethyl]-methyl-carbamoyl}-2-(1H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester (Example 2). Coupling of Boc-NMe-Tyr(OBn)-OH to2-phenethylamine was carried out in methylene chloride:dimethylformamide(DMF) (1:1) with dicyclohexylcarbodiimide (DCC) and1-hydroxybenzotriazole (HOBt), as coupling agents. The resulting productwas treated with 25% trifluoroacetic acid (TFA) in methylene chloride.H-NMe-Tyr(OBn)-NH—CH₂—CH₂-phenyl. TFA was then coupled in methylenechloride:DMF (1:1) to Cbz-His(Trt)-OH with (O-(7-azabenzotriazol-1-yl)1,1,3,3-tetramethyl uronium hexafluorophosphate (HATU) and1-hydroxy-7-azabenzotriazole (HOAt) as coupling agents, anddiisopropylethylamine (DIEA) as the base, followed by treatment with50%TFA in methylene chloride to give the title compound.

Scheme 3 illustrates a general method by which some of these compoundscan be prepared, by illustrating the synthesis of[S-(R*,R*)]-[1-{[2-(4-benzyloxy-phenyl)-1-(2-methyl-2-phenyl-propylcarbarmoyl)-ethyl]-methyl-carbamoyl}-2-(1H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester (Example 3). Coupling of Boc-NMe-Tyr(OBn)-OH toβ,β-dimethylphenethylamine hydrochloride was carried out in methylenechloride:DMF (3:1) with dicyclohexylcarbodiimide (DCC) and1-hydroxybenzotriazole (HOBt), as coupling agents, and DIEA as the base.The resulting product was treated with 25% trifluoroacetic acid (TFA) inmethylene chloride. H-NMe-Tyr(OBn)-NH—CH₂—C(CH₃)₂-phenyl. TFA was thencoupled in methylene chloride to Cbz-His(Trt)-OH, withbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP) as coupling agent, and diisopropylethylamine (DIEA) as the base,followed by treatment with 50%TFA in methylene chloride to give thetitle compound.

β,β-Dimethylphenethylamine hydrochloride was obtained from benzylcyanide, which was treated with 2 equivalents of sodium hydride intetrahydrofuran (THF) and 2 equivalents of methyl iodide in THF,followed by hydrogenation (H₂, Pd/C, ammonia/CH₃OH). The product wasisolated as the hydrochloride salt.

Scheme 4 illustrates a general method by which some of these compoundscan be prepared, by illustrating the synthesis of[S-(R*,R*)]-[2-(1H-imidazol-4-yl)-1-(methyl-{1-(2-methyl-2-phenyl-propylcarbamoyl)-2-[4-(pyridin-2-ylmethoxy)-phenyl]-ethyl}-carbamoyl)-ethyl]-carbamicacid benzyl ester (Example 4). Coupling of Boc-NMe-Tyr-OH toβ,β-dimethylphenethylamine hydrochloride was carried out in THF withdicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt), ascoupling agents, and triethylamine as the base.Boc-NMe-Tyr-NH—CH₂-C(CH₃)₂-phenyl was dissolved in THF and treated with2-pyridylcarbinol and triphenylphosphine followed by diethylazodicarboxylate, under a nitrogen atmosphere. The resulting product wastreated with 33% trifluoroacetic acid (TFA) in methylene chloride.H-NMe-Tyr(O—CH₂-(2-pyridyl))-NH—CH₂—C(CH₃)₂-phenyl. TFA was then coupledin methylene chloride to Cbz-His(Trt)-OH, withbenzotriazole-1-yl-oxy-tris-pyrrolidino phosphonium hexafluorophosphate(PyBOP) as coupling agent, and diisopropylethylamine (DIEA) as the base,followed by treatment with 80% aqueous acetic acid (HOAc) at 87° C. togive the title compound.

Scheme 5 illustrates a general method by which some of these compoundscan be prepared, by illustrating the synthesis of[S-(R*,R*)]-2-(3-benzyl-3-methyl-ureido)-N-[2-(4-benzyloxy-phenyl)-1-(2-methyl-2-phenyl-propylcarbarmoyl)-ethyl]-3-(1H-imidazol-4-yl)-N-methyl-propionamide(Example 5). Coupling of Boc-NMe-Tyr(OBn)-OH toβ,β-dimethylphenethylamine hydrochloride was carried out in chloroformwith benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate (PyBOP) as coupling agent, and triethylamine as thebase. The resulting product was then treated with a saturated solutionof HCl in diethyl ether to give H-NMe-Tyr(OBn)-NH—CH₂—C(CH₃)₂-phenyl.HCl(B).

H-His(Trt)-OCH₃ hydrochloride was reacted with4-nitro-phenyl-chloroformate in the presence of triethylamine inmethylene chloride, followed by the addition of benzylmethylamine.Saponification was then carried out with 1N NaOH in methanol:THF (1:1)followed by treatment with 1N HCl to givephenyl-CH₂—N(CH₃)—CO-His(Trt)-OH (A).

Products A and B were then coupled in chloroform withbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP) as coupling agent, and DIEA as the base. The resulting productwas then treated with 80% aqueous acetic acid at 90° C. to give thetitle compound.

Scheme 6 illustrates a general method by which some of these compoundscan be prepared, by illustrating the synthesis of[S-(R*,R*)]-[1-({2-(4-benzyloxy-phenyl)-1-[(1-phenyl-cyclobutylmethyl)-carbamoyl]-ethyl}-methyl-carbamoyl)-2-(3H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester (Example 6). Coupling of Boc-NMe-Tyr(OBn)-OH toC-(1-phenyl-cyclobutyl)-methylamine hydrochloride was carried out inmethylene chloride with2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) as coupling agent, and DIEA as the base. The resulting productwas treated with 20% trifluoroacetic acid (TFA) in methylene chloride.(S)-3-(4-Benzyloxy-phenyl)-2-methylamino-N-(1-phenyl-cyclobutylmethyl)-propionamidetrifluoroacetate salt was then coupled in methylene chloride toCbz-His(Trt)-OH with HBTU as coupling agent, and diisopropylethylamine(DIEA) as the base, followed by treatment with 50%TFA in methylenechloride to give the title compound.

Scheme 7 illustrates a general method by which some of these compoundscan be prepared, by illustrating the synthesis of[S-(R*,R*)]-(2-(3H-imidazol-4-yl)-1-{methyl-[1-(2-methyl-2-phenyl-propylcarbamoyl)-2-phenyl-ethyl]-carbamoyl}-ethyl)-carbamicacid benzyl ester (Example 7). Coupling of Boc-NMe-Phe-OH toβ,β-dimethylphenethylamine hydrochloride was carried out in methylenechloride with dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole(HOBt), as coupling agents, and DIEA as the base. The resulting productwas treated with 30% trifluoroacetic acid (TFA) in methylene chloride.H-NMe-Phe-NH—CH₂—C(CH₃)₂-phenyl.TFA was then coupled in methylenechloride to Cbz-His(Trt)-OH with HATU and HOAt as coupling agents anddiisopropylethylamine (DIEA) as the base, followed by treatment with50%TFA in methylene chloride to give the title compound.

Scheme 8 illustrates a general method by which some of these compoundscan be prepared, by illustrating the synthesis of[S-(R*,R*)]-(2-(3H-imidazol-4-yl)-1-{methyl-[3-methyl-1-(2-methyl-2-phenyl-propylcarbamoyl)-butyl]-carbamoyl}-ethyl)-carbamicacid benzyl ester (Example 8). Coupling of Boc-NMe-Leu-OH toβ,β-dimethylphenethylamine hydrochloride was carried out in methylenechloride:DMF (1:1) with HBTU as coupling agent, and DIEA as the base.The resulting product was treated with 25% trifluoroacetic acid (TFA) inmethylene chloride. H-NMe-Leu-NH—CH₂—C(CH₃)₂-phenyl.TFA was then coupledin methylene chloride:DMF (4:1) to Cbz-His(Trt)-OH, with HBTU ascoupling agent, and diisopropylethylamine (DEA) as the base, followed bytreatment with 50%TFA in methylene chloride to give the title compound.

The following abbreviations are used herein:

Abbreviations Cbz or Z Carbobenzoxy His Histidine Trt Trityl TEATriethylamine HOAc Acetic acid Et₂O Diethylether tBu tert-Butyl TFATrifluoroacetic acid ES-MS Electrospray Mass Spectrometry FAB-MS FastAtom Bombardment Mass Spectrometry HOBt 1-Hydroxybenzotriazole DCCDicyclohexylcarbodiimide THF Tetrahydrofuran PyBOPBenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphateDIEA Diisopropylethylamine DMF Dimethylformamide Et₃N Triethylamine OAcAcetate Et₂O Diethyl ether Boc tert-Butoxycarbonyl iBuOCOClIsobutylchloroformate NMM N-methylmorpholine DMSO Dimethylsulfoxide HATU(O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate HOAt 1-Hydroxy-7-azabenzotriazole HBTU2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphateEtOAc Ethylacetate PPh₃ Triphenyl phosphine Hepes(N-[Z-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid])

EXAMPLE 1[R-(R*,S*)]-[1-{[1-(2-Benzyloxy-ethylcarbamoyl)-2-(4-benzyloxy-phenyl)-ethyl]-methyl-carbamoyl}-2-(1H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester

Step 1

Boc-NMe-Tyr(OBn)-NH—CH₂CH₂-OBn or(S)-[1-(2-Benzyloxy-ethylcarbamoyl)-2-(4-benzyloxy-phenyl)-ethyl]-methyl-carbamicacid tert-butyl ester

To a solution of Boc-NMe-Tyr(OBn)-OH (0.82 g, 2.1 mmol) in ethyl acetate(100 mL) at 0° C. was added HOBt (0.40 g, 2.95 mmol) followed by DCC(0.53 g, 2.58 mmol) and 2-(phenylmethoxy)-ethylamine hydrochloride (fromStep 5, below; 0.40 g, 2.1 mmol), followed by triethylamine (0.31 mL,2.2 mmol). The mixture was allowed to warm to room temperature andstirred overnight. The mixture was filtered, diluted with ethyl acetate,and washed twice with 2N HCl, 1N NaHCO₃, brine, dried over MgSO₄,filtered, and concentrated. The product was purified by flashchromatography (1:1 ethyl acetate:hexanes) (0.90 g, 1.7 mmol; 81%);MS-CI 519 (m+1).

Step 2

HNMe-Tyr(OBn)-NH—CH₂—CH₂-OBn.TFA or(S)-N-(2-Benzyloxy-ethyl)-3-(4-benzyloxy-phenyl)-2-methylamino-propionamide.TFA

Boc-NMe-Tyr(OBn)-NH—CH₂—CH₂-OBn (from Step 1, 0.90 g, 1.7 mmol) wastreated with 50 mL of 30% trifluoroacetic acid (TFA) in methylenechloride. The solution was stirred at room temperature for 4 hours. Thesolution was concentrated, the residue taken up in methylene chlorideand reconcentrated. This was repeated twice. The residue was dried invacuo and used without further purification; MS-CI 419 (m+1).

Step 3

Cbz-DHis(Trt)-NMe-Tyr(OBn)-NH—CH₂—CH₂-OBn or[S-(R*,S*)]-[1-[1-(2-Benzyloxy-ethylcarbamoyl)-2-(4-benzyloxy-phenyl)-ethylcarbamoyl]-2-(1-trityl-1H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester

To a solution of Cbz-DHis(Trt)-OH (synthesized according to the methodin Hudspeth J. P., Kaltenbronn J. S., Repine J. T., Roark W. H., StierM. A., Renin inhibitors III, U.S. Pat. No. 4,735,933; 1988) (0.94 g, 1.8mmol) in methylene chloride (50 mL) was addedbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP; 0.93 g, 1.8 mmol). HNMe-Tyr(OBn)-NH—CH₂—CH₂-OBn. TFA (from Step2, 1.7 mmol) was added followed by diisopropylethylamine (DIEA; 0.76 mL,4.4 mmol). The mixture was stirred overnight at room temperature. Thesolution was concentrated in vacuo, and the residue was taken up inethyl acetate. The organic solution was washed twice with saturatedNaHCO₃, brine, dried over MgSO₄, filtered, and concentrated (1.55 g,1.66 mmol); MS-ES 933 (m+1).

Step 4

Cbz-DHis-NMe-Tyr(OBn)-NH—CH₂—CH₂-OBn or{R-(R*,S*)]-[1-{[1-(2-Benzyloxy-ethylcarbamoyl)-2-(4benzyloxy-phenyl)-ethyl]-methyl-carbamoyl}-2-(1H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester

Cbz-DHis(Trt)-NMe-Tyr(OBn)-NH—CH₂—CH₂-OBn (from Step 3, 1.55 g, 1.66mmol) was treated with 50 mL of 50% TFA in methylene chloride. Thesolution was stirred at room temperature for 3 hours. The solution wasconcentrated, the residue taken up in methylene chloride, andreconcentrated. This was repeated twice. The residue was dried in vacuo.Purification was carried out on a portion of the product (0.2 g) byreverse-phase preparative HPLC (C-18 reverse-phase column, 20% to 65% of0.1% TFA in acetonitrile against 0.1%TFA in water, 100 minutes, 13mL/min) to give 18.5 mg of a white foam; MS-ES 690.3 (m+1).

CHN: Calculated for C₄₀H₄₃N₅O₆.1.48CF₃COOH.0.49H₂O C, 59.46; H, 5.28; N,8.07.

Found: C, 59.46; H, 5.28; N, 8.12.

Step 5

2-(Phenylmethoxy)-ethylamine hydrochloride

Ethanolamine hydrochloride (20 g, 0.21 mol) in toluene (100 mL) wastreated with sodium metal (pellets washed with hexane) (10.14 g, 0.44mL). The reaction was refluxed until the sodium metal was no longerpresent. The mixture was cooled and benzyl chloride (24.17 mL, 0.21 mol)was added, the reaction was stirred at room temperature overnight. Thereaction mixture was filtered and the solid washed with toluene. Thefiltrate was cooled to 0° C. and HCl gas was bubbled in for 10 minutes.A white precipitate was obtained and filtered. The solid wasrecrystallized using 70 mL of isopropyl alcohol (10.4 g, 0.056 mol);MS-CI 188 (m+1).

EXAMPLE 2[S-(R*,R*)]-[1-{[2-(4-Benzyloxy-phenyl)-1-phenethylcarbamoyl-ethyl]-methyl-carbamoyl}-2-(1H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester

Step 1

Boc-NMe-Tyr(OBn)-NH—CH₂—CH₂-phenyl or(S)-[2-(4-Benzyloxy-phenyl)-1-phenethylcarbamoyl-ethyl]-methyl-carbamicacid tert-butyl ester

To a solution of Boc-NMe-Tyr(OBn)-OH (0.77 g, 2.0 mmol) in methylenechloride:dimethylformamide (1:1; 20 mL) was added HOBt (0.30 g, 2.2mmol) followed by 0.5 M DCC/CH₂Cl₂ (4.4 mL g, 2.2 mmol). After stirring1 hour, phenethylamine (0.27 mL, 2.2 mmol) was added. The mixture wasstirred overnight at room temperature. The mixture was filtered and thesolvent removed in vacuo. The residue was taken up in ethyl acetate andwashed twice saturated NaHCO₃, brine, dried over MgSO₄, filtered, andconcentrated. The product was used without further purification.

Step 2

HNMe-Tyr(OBn)-NH—CH₂—CH₂-phenyl.TFA or(S)-3-(4-Benzyloxy-phenyl)-2-methylamino-N-phenethyl-propionanide.TFA

Boc-NMe-Tyr(OBn)-NH—CH₂—CH₂-phenyl (from Step 1, 2.0 mmol) was treatedwith 50 mL of 25% trifluoroacetic acid (TFA) in methylene chloride. Thesolution was stirred at room temperature for 4 hours. The solvent wasreduced in volume and cold diethyl ether was added to precipitate theproduct (0.87 g, 2 mmol); MS-CI 389 (m+1).

Step 3

Cbz-His(Trt)-NMe-Tyr(OBn)-NH—CH₂—CH₂-phenyl or[S-(R*,S*)]-[1-[1-(2-Benzyloxy-ethylcarbamoyl)-2-(4-benzyloxy-phenyl)-ethylcarbamoyl]-2-(1-trityl-1H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester

To a solution of Cbz-His(Trt)-OH (synthesized according to the method inHudspeth J. P., Kaltenbronn J. S., Repine J. T., Roark W. H., Stier M.A., Renin inhibitors III, U.S. Pat. No. 4,735,933; 1988) (0.38 g, 0.7mmol) in methylene chloride (5 mL) was added(O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate (HATU; 0.149 g, 0.4 mmol) and1-hydroxy-7-azabenzotriazole (HOAt; 0.063 g, 0.5 mmol), dissolved indimethylformamide (5 mL). HNMe-Tyr(OBn)-NH—CH₂—CH₂-phenyl.TFA (from Step2, 0.25 g, 0.64 mmol) was added followed by diisopropylethylamine (DIEA;0.275 mL, 1.6 mmol). The mixture was stirred overnight at roomtemperature. The solution was concentrated in vacuo, and the residue wastaken up in ethyl acetate. The organic solution was washed twice withsaturated NaHCO₃, brine, dried over MgSO₄, filtered, and concentrated togive 0.5 g of product (0.55 mmol), which was used without furtherpurification.

Step 4

Cbz-His-NMe-Tyr(OBn)-NH—CH₂—CH₂-Phenyl or[S-(R*,R*)]-[1-{[2-(4-Benzyloxy-phenyl)-1-phenethylcarbamoyl-ethyl]-methyl-carbamoyl}-2-(1H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester

Cbz-His(Trt)-NMe-Tyr(OBn)-NH—CH₂—CH₂-OBn (from Step 3, 0.5 g, 0.55 mmol)was treated with 50 mL of 50% TFA in methylene chloride. The solutionwas stirred at room temperature for 2 hours. The solvent was reduced involume and diethyl ether/hexane (200 mL) was added to the residue. Thesolution was cooled to −40° C. overnight. The ether/hexane was decantedand the residue dried in vacuo. Purification was carried out byreverse-phase preparative HPLC (C-18) reverse-phase column, 20% to 60%of 0.1% TFA in acetonitrile against 0.1% TFA in water, 100 minutes, 13mL/min) to give 37.5 mg of a white foam;

MS-ES 661 (m+1).

CHN: Calculated for C₃₉H₄₂N₅O₅.1.37CF₃COOH.0.08H₂O C, 61.25; H, 5.36; N,8.56.

Found: C, 61.25; H, 5.36; N, 8.55.

EXAMPLE 3[S-(R*,R*)]-[1-{[2-(4-Benzyloxy-phenyl)-1-(2-methyl-2-phenyl-propylcarbamoyl)-ethyl]-methyl-carbamoyl}-2-(1H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester

Step 1

Boc-NMe-Tyr(OBn)-NH—CH₂—C(CH₃)₂-phenyl or(S)-[2-(4-Benzyloxy-phenyl)-1-(2-methyl-2-phenyl-propylcarbamoyl)-ethyl]-methyl-carbamicacid tert-butyl ester

To a solution of Boc-NMe-Tyr(OBn)-OH (1.54 g, 4.0 mmol) in methylenechloride:dimethylformamide (3:1; 20 mL) was added HOBt (0.60 g, 4.4mmol) followed by 0.5 M DCC/CH₂Cl₂ (8.8 mL g, 4.4 mmol).β,β-Dimethyl-phenethylamine hydrochloride (from Step 5 below) (0.815 g,4.4 mmol) and DIEA (0.80 mL, 4.6 mmol) were then added. The mixture wasstirred overnight at room temperature. The mixture was filtered and thesolvent removed in vacuo. The residue was taken up in ethyl acetate andwashed twice saturated NaHCO₃, brine, dried over MgSO₄, filtered, andconcentrated (1.81 g, 3.5 mmol; 87% yield); MS-APCI 518 (m+1).

Step 2

HNMe-Tyr(OBn)-NH—CH₂—C(CH₃)₂-phenyl.TFA or(S)-3-(4-Benzyloxy-phenyl)-2-methylamino-N-(2-methyl-2-phenyl-propyl)-propionamide.TFA

Boc-NMe-Tyr(OBn)-NH—CH₂—(CH₃)₂-phenyl (from Step 1, 3.5 mmol) wastreated with 50 mL of 25% TFA in methylene chloride. The solution wasstirred at room temperature for 2 hours. The solvent was reduced involume and the residue was added to 1:1 hexanes:diethyl ether, which wasstored overnight at −40° C. overnight to precipitate the product. Theether/hexane was decanted and the residue dried in vacuo (1.2 g, 2.8mmol); MS-CI 389 (m+1). The product was used without furtherpurification.

Step 3

Cbz-His(Trt)-NMe-Tyr(OBn)-NH—CH₂—C(CH₃)₂-phenyl or[S-(R*,R*)]-[1-{[2-(4-Benzyloxy-phenyl)-1-(2-methyl-2-phenyl-propylcarbamoyl)-ethyl]-methyl-carbamoyl}-2-(1-trityl-1H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester

To a solution of Cbz-His(Trt)-OH (synthesized according to the method inHudspeth J. P., Kaltenbronn J. S., Repine J. T., Roark W. H., Stier M.A., Renin inhibitors III, U.S. Pat. No. 4,735,933; 1988) (0.64 g, 1.2mmol) in methylene chloride (5 mL) was addedbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP; 0.62 g, 1.2 mmol). HNMe-Tyr(OBn)-NH—CH₂—C(CH₃)₂-phenyl.TFA (fromStep 2, 0.58 g, 1.2 mmol) was added followed by diisopropylethylamine(DIEA; 0.545 mL, 3.3 mmol). The mixture was stirred overnight at roomtemperature. The solution was concentrated in vacuo, and the residue wastaken up in ethyl acetate. The organic solution was washed twice withsaturated NaHCO₃, brine, dried over MgSO₄, filtered, and concentrated.The product was used without further purification.

Step 4

Cbz-His-NMe-Tyr(OBn)-NH—CH₂—C(CH₃)₂-phenyl or[S-(R*,R*)]-[1-{[2-(4-Benzyloxy-phenyl)-1-(2-methyl-2-phenyl-propylcarbamoyl)-ethyl]-methyl-carbamoyl}-2-(1H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester Cbz-His(Trt)-NMe-Tyr(OBn)-NH—CH₂—C(CH₃)₂-phenyl (fromStep 3, 1.2 mmol) was treated with 50 mL of 50% TFA in methylenechloride. The solution was stirred at room temperature for 2 hours. Thesolvent was reduced in volume and the residue was added to 1:1hexanes:diethyl ether, which was stored overnight at −40° C. overnightto precipitate the product. The ether/hexane was decanted and theresidue dried to vacuo. Purification was carried out by reverse-phasepreparative HPLC (C-18) reverse-phase column, 20% to 60% of 0.1% TFA inacetonitrile against 0.1% TFA in water, 100 minutes, 13 mL/min) to give25 mg of a white foam (4%); MS-ES 688 (m+1).

CHN: Calculated for C₄₁H₄₅N₅O₅.1.11CF₃COOH.1.67H₂O C, 61.44; H, 5.90; N,8.29.

Found: C, 61.44; H, 5.90; N, 8.11.

Step 5

β,β-Dimethylphenethylamine hydrochloride

Sodium hydride (60% in oil) (17 g, 0.43 mol) was suspended in THF (150mL) and cooled to 0° C. under nitrogen. Benzyl cyanide (22.2 g, 0.19mol) in THF (30 mL) was added dropwise and the reaction was left to stirfor 1 hour. Iodomethane (24.9 mL, 0.4 mmol) in THF (20 mL) was addeddropwise at 0° C. The reaction was stirred at room temperatureovernight, under nitrogen. The solution was filtered, and the filtrateremoved in vacuo. The residue was taken up in ethyl acetate (100 mL) andwashed 3 times with 10% NaHSO₃, saturated NaHCO₃, brine, dried overMgSO₄, and concentrated; 22.74 g (92%).

Reduction of the above product was carried out in the presence of Raneynickel, in methanol/NH₃. The catalyst was removed and washed withmethanol. The filtrate was concentrated and diethyl ether (100 mL) andadded to the residue. Concentrated HCl was added dropwise to precipitatethe desired product; 24.8 g (86%).

EXAMPLE 4[S-(R*,R*)]-[2-(1H-Imidazol-4-yl)-1-(methyl-{1-(2-methyl-2-phenyl-propylcarbamoyl)-2-[4-(pyridin-2-ylmethoxy)-phenyl]-ethyl}-carbamoyl)-ethyl]-carbamicacid benzyl ester

Step 1

Boc-NMe-Tyr-NH—CH₂—C(CH₃)₂-phenyl

A solution of Boc-NMe-Tyr-OH (3.87 g, 13.1 mmol) in tetrahydrofuran(THF) (70 mL) was treated with β,β-dimethylphenethylamine hydrochloride(from Step 5, Example 3) (2.44 g, 13.1 mmol), HOBt (1.78 g, 13.1 mmol)and DCC (2.74 g, 13.1 mmol). Triethylamine (1.9 mL, 13.1 mmol) was thenadded and the mixture stirred at room temperature for 2 days. A solutionof 1N citric acid (26 mL, 26 mmol) was then added and the mixturefiltered. The filtrate was diluted with ethyl acetate and washed with 1Ncitric acid, water, and brine. It was dried over MgSO₄ and removal ofthe solvent under reduced pressure gave the crude product (6.85 g). Onadding chloroform/ethyl acetate, the HOBt precipitated and it wasremoved by filtration. The solvent was removed under reduced pressureand the residue chromatographed (CHCl₃:CH₃OH 98:2) to give the product(4.32 g, 8.4 mmol; 77% yield); MS-APCI 427 (m+1).

Step 2

Boc-Nme-Tyr-(O—CH₂)-(2-pyridyl))-NH—CH₂—C(CH₃)₂-phenyl or(S)-Methyl-{1-(2-methyl-2-phenyl-propylcarbamoyl)-2-[4-(pyridin-2-ylmethoxy)-phenyl]-ethyl}-carbamicacid tert-butyl ester

Under nitrogen atmosphere, a solution ofBoc-NMe-Tyr-NH—CH₂—C(CH₃)₂-phenyl (from Step 1) (4.32 g, 10.1 mmol) inTHF (50 mL) was treated with 2-pyridylcarbinol (1.1 mL, 11.1 mmol) andtriphenylphosphine (2.69 g, 10.1 mmol). The solution was cooled in iceand treated dropwise over 20 minutes, with diethyl azodicarboxylate (1.6mL, 10.1 mmol) in THF (5 mL). The cooling was removed and the solutionallowed to stir at room temperature for 3 days. The solution was dilutedwith ethyl acetate and washed 3 times with water, brine, dried overMgSO₄ and the solvent removed in vacuo (10.8 g). The residue waschromatographed (CHCl₃:CH₃OH 98:2) to give the product (4.38 g, 8.4mmol; 84% yield); MS-APCI 518 (m+1).

Step 3

HNMe-Tyr(O—CH₂-(2-pyridyl))-NH—CH₂—C(CH₃)₂-phenyl or(S)-2-Methylamino-N-(2-methyl-2-phenyl-propyl)-3-[4-(pyridin-2-ylmethoxy)-phenyl]-propionamide.TFA

A solution of Boc-NMe-Tyr(O—CH₂-(2-pyridyl))-NH—CH₂—C(CH₃)₂-phenyl (fromStep 2, 1.8 g, 3.5 mmol) in methylene chloride (20 mL) was treated withtrifluoroacetic acid (10 mL) and allowed to stir at room temperature for1 hour. The solvent was removed under reduced pressure and then theresidue was taken up in ethyl acetate. This solution was washed twicewith saturated NaHCO₃, brine, dried over MgSO₄, and the solvent removedunder reduced pressure to give the product (1.39 g, 3.3 mmol); MS-APCI418 (m+1).

Step 4

Cbz-His(Trt)-NMe-Tyr(O—CH₂-(2-pyridyl))-NH—CH₂—C(CH₃)₂-phenyl or[S-(R*,R*)]-[1-(Methyl-{1-(2-methyl-2-phenyl-propylcarbamoyl)-2-[4-(pyridin-2-ylmethoxy)-phenyl]-ethyl}-carbamoyl)-2-(1-trityl-1H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester

A solution of HNMe-Tyr(O CH₂-(2-pyridyl))-NH—CH₂—C(CH₃)₂-phenyl (fromStep 3) (1.39 g, 3.3 mmol) in methylene chloride (50 mL) was treatedwith Cbz-His-(Trt)-OH (synthesized according to the method in HudspethJ. P., Kaltenbronn J. S., Repine J. T., Roark W. H., Stier M. A., Renininhibitors III, U.S. Pat. No. 4,735,933; 1988) (1.78 g, 3.3 mmol). Thesolution was cooled in ice and treated with DIEA (1.8 mL, 9.9 mmol),followed by PyBOP (1.75 g, 3.3 mmol). The cooling was removed and thesolution stirred at room temperature for 2 days. The solvent was removedin vacuo and the residue taken up in ethyl acetate, and washed twicewith water and brine. Drying over MgSO₄ and removal of the solvent underreduced pressure gave the crude product. It was taken up in a smallamount of ethyl acetate, and filtered. Removal of the solvent underreduced pressure gave 3.39 g of the crude product. Chromatography(CHCl₃:CH₃OH 98:2) gave the desired product (2.01 g, 2.2 mmol; 65%yield); MS-ES 932 (m+1).

Step 5

Cbz-His-NMe-Tyr(O—CH₂-(2-pyridyl))-NH—CH₂—C(CH₃)₂-phenyl or[S-(R*,R*)]-[2-(1H-Imidazol-4-yl)-1-(methyl-{1-(2-methyl-2-phenyl-propylcarbamoyl)-2-[4-pyridin-2-ylmethoxy)-phenyl]-ethyl}-carbamoyl)-ethyl]-carbamicacid benzyl ester

A solution ofCbz-His(Trt)-NMe-Tyr(O—CH₂-(2-pyridyl))-NH—CH₂-C(CH₃)₂-phenyl (from Step4, 1.49 g, 1.6 mmol) in 80% acetic acid/water (100 mL) was heated at 87°C. for 30 minutes. The solvent was removed under reduced pressure andthe residue taken up in ethyl acetate, and washed twice with saturatedNaHCO₃ and brine. Drying over MgSO₄ and removal of the solvent underreduced pressure gave the crude product. Chromatography (CHCl₃:CH₃OH95:5) gave the product. It was dissolved in methylene chloride and thesolvent removed under reduced pressure to give the desired product (0.82g, 1.2 mmol; 74% yield); MS-APCI 690 (m+1).

CHN: Calculated for C₄₀H₄₄N₆O₅.0.2CH₂Cl₂ C, 68.41; H, 6.34; N, 11.91.

Found: C, 68.59; H, 6.32; N, 11.87.

EXAMPLE 5[S-(R*,R*)]-2(3-Benzyl-3-methyl-ureido)-N-[2-(4-benzyloxy-phenyl)-1-(2-methyl-2-phenyl-propyl-carbamoyl)-ethyl]-3-(1H-imidazol-4-yl)-N-methyl-propionamide

Step 1

Phenyl-CH₂—N(CH₃)—CO-His(Trt)-OCH₃ or(S)-2-(3-Benzyl-3-methyl-ureido)-3-(1-trityl-1H-imidazol-4-yl)-propionicacid methyl ester

Histidine-(trityl)-methyl ester hydrochloride (20 g, 0.042 mol) wassuspended in methylene chloride (300 mL) and washed twice with saturatedNaHCO₃, brine, dried over MgSO₄, filtered, and cooled to 0° C.Triethylamine (6.54 mL, 0.047 mol) and 4-nitro-phenyl-chloroformate(9.38 g, 0.047 mol) were added and the solution stirred for 1 hour.Benzylmethylamine (11.4 mL, 0.088 mol) in methylene chloride (100 mL)was then added dropwise. The reaction was stirred at room temperaturefor 72 hours. The solvent was removed in vacuo. Ethyl acetate was addedto the residue, and it was washed 3 times with saturated NaHCO₃, water,and brine. The solution was dried over MgSO₄, filtered, and concentratedin vacuo. The oil was chromatographed (CHCl₃ to CHCl₃:CH₃OH 96:4) togive the desired product (11.55 g, 0.021 mol; 49% yield); MS-APCI 559(m+1).

Step 2

Phenyl-CH₂—N(CH₃)—CO-His(Trt)-OH or(S)-2-(3-Benzyl-3-methyl-ureido)-3-(1-trityl-1H-imidazol-4-yl)-propionicacid

Phenyl-CH₂—N(CH₃)—CO-His(Trt)-OCH₃ (from Step 1; 6.66 g, 0.012 mol) wasdissolved in CH₃OH:THF (50 mL each). 1N NaOH (36 mL, 0.036 mol) wasadded dropwise and the solution was stirred at room temperature for 1.5hours. The solvent was removed in vacuo. 1N HCl (36 mL, 0.036 mol) wasadded to the residue. The product was extracted with ethyl acetate. Theorganic solution was then washed with brine, dried over MgSO₄, filtered,and concentrated in vacuo. The solid product crystallized (5.83 g, 0.011mol; 89% yield); MS-APCI 545 (m+1).

Step 3

Boc-NMe-Tyr(OBn)-NH—CH₂—C(CH₃)₂-phenyl or(S)-[2-(4-Benzyloxy-phenyl)-1-(2-methyl-2-phenyl-propylcarbamoyl)-ethyl]-methyl-carbamicacid tert-butyl ester

Boc-NMe-Tyr(OBn)-OH (0.83 g, 2.1 mmol), β,β-dimethylphenethylaminehydrochloride (from Step 5, Example 3) (0.44 g, 2.4 mmol), and DIEA(0.84 mL, 4.8 mmol) were dissolved in chloroform (20 mL) and cooled at0° C., under nitrogen. After 15 minutes, PyBOP (1.25 g, 2.4 mmol) wasadded and the reaction was stirred at room temperature for 48 hours. Thesolvent was removed in vacuo and ethyl acetate was added to the residue.The organic solution was washed twice with water, saturated NaHCO₃,brine, dried over MgSO₄, filtered, and concentrated in vacuo. The oilobtained was purified by chromatography (ethyl acetate:hexane 70:30)(1.08 g, 2.1 mmol; 100% yield); MS-APCI 517 (m+1).

Step 4

H NMe-Tyr(OBn)-NH—CH₂—C(CH₃)₂-phenyl or(S)-3-(4-Benzyloxy-phenyl)-2-methylamino-N-(2-methyl-2-phenyl-propyl)-propionamide.TFA

Boc-NMe-Tyr(OBn)-NH—CH₂—C(CH₃)₂-phenyl (from Step 3, 1.04 g, 2 mmol) wasdissolved in diethyl ether saturated with HCl. The reaction was stirredovernight at room temperature. The solvent was removed in vacuo to givethe product which was used without further purification. (1.0 g, 2.4mmol); MS-APCI 417 (m+1).

Step 5

Phenyl-CH₂—N(CH₃)—CO-His(Trt)-NMe-Tyr(OBn)-NH—CH₂—C(CH₃)₂-phenyl or[S-(R*,R*)]-2-(3-Benzyl-3-methyl-ureido)-N-[2-(4-benzyloxy-phenyl)-1-(2-methyl-2-phenyl-propylcarbamoyl)-ethyl]-N-methyl-3-(1-trityl-1H-imidazol-4-yl)-propionamide

Phenyl-CH₂—N(CH₃)—CO-His(Trt)-OH (from Step 2; 1.09 g, 2.0 mmol),H-NMe-Tyr(OBn)-NH—CH₂—C(CH₃)₂-phenyl (from Step 4; 0.80 g, 1.8 mmol) andDIEA (0.71 mL, 4.1 mmol) were dissolved in chloroform (20 mL) and cooledto 0° C., under nitrogen. After 15 minutes, PyBOP (1.04 g, 2.0 mmol) wasadded and the reaction was stirred at room temperature overnight. Thesolvent was removed in vacuo and ethyl acetate was added to the residue.The organic solution was washed twice with water, saturated NaHCO₃,brine, dried over MgSO₄, filtered, and concentrated in vacuo. Theresidue obtained was purified by chromatography (ethyl acetate:hexane70:30) (1.39 g, 1.5 mmol; 85% yield); MS-APCI 943 (m+1).

Step 6

Phenyl-CH₂—N(CH₃)—CO-His-NMe-Tyr(OBn)-NH—CH₂—C(CH₃)₂-phenyl or[S-(R*,R*)]-2-(3-Benzyl-3-methyl-ureido)-N-[2-(4-benzyloxy-phenyl)-1-(2-methyl-2-phenyl-propylcarbamoyl)-ethyl]-3-(1H-imidazol-4-yl)-N-methyl-propionamide

Phenyl-CH₂—N(CH₃)—CO-His(Trt)-NMe-Tyr(OBn)-NH—CH₂—C(CH₃)₂-phenyl (fromStep 5; 1.39 g, 1.5 mmol) was dissolved in acetic acid:water (5:1, 12mL) and heated to 90° C. for 30 minutes. The solvent was removed invacuo. Ethyl acetate was added to the residue and washed twice withsaturated NaHCO₃, a brine, dried over MgSO₄, filtered, and concentratedin vacuo. The oil obtained was chromatographed (CHCl₃ to CHCl₃:CH₃OH94:6) (0.34 g 0.5 mmol; 32% yield); MS-APCI 701 (m+1).

CHN: Calculated for C₄₂H₄₈N₆O₄.0.27CHCl₃.0.32H₂O C, 68.71; H, 6.67; N,11.37.

Found: C, 68.71; H, 6.71; N, 11.23.

EXAMPLE 6[S-(R*,R*)]-[1-{[2-4-Benzyloxy-phenyl)-1-[(1-phenyl-cyclobutylmethyl)-carbamoyl]-ethyl}-methyl-carbamoyl)-2-(3H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester

Step 1

(S)-{2-(4-Benzyloxy-phenyl)-1-[(1-phenyl-cyclobutylmethyl)-carbamoyl]-ethyl}-methyl-carbamicacid tert-butyl ester

A suspension of Boc-NMe-Tyr(OBn)-OH (0.90 g, 2.3 mmol) andC-(1-phenyl-cyclobutyl)-methylamine hydrochloride (synthesized accordingto the method in Bridges, A. J., Hamilton, H. W., Moos, W. H., Szotek,D. L., N⁶⁻substituted Adenosines, U.S. Pat. No. 4,755,594) (0.46 g, 2.3mmol) and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) (0.97 g, 5.6 mmol) in methylene chloride (10mL) was treated with DIEA (0.86 mL, 5.1 mmol). The suspension wasstirred at room temperature overnight. The resulting solution wasconcentrated. The residue was taken up in ethyl acetate. The solutionwas washed with 0.5 M HCl, saturated NaHCO₃, dried over MgSO₄, filtered,and concentrated. The oil was chromatographed (CH₂Cl₂:CH₃OH 50:1) togive the desired product (1.07 g, 2.02 mmol; 87% yield). MS-APCI 529(m+1).

Step 2

(S)-3-(4-Benzyloxy-phenyl)-2-methylamino-N-(1-phenyl-cyclobutylmethyl)-propionamidetrifluoroacetate salt

To a solution of(S)-{2-(4-Benzyloxy-phenyl)-1-[(1-phenyl-cyclobutylmethyl)-carbamoyl]-ethyl}-methyl-carbamicacid tert-butyl ester (from Step 1; 1.07 g, 2.02 mmol) in methylenechloride (10 mL) was added TFA (2.5 mL). The solution was stirred atroom temperature for 2 hours and then concentrated in vacuo.Coevaporisation with methylene chloride was carried out twice to givethe desired product which was used without further purification.

Step 3

[S-[R*,R*)]-[1-({2-(4-Benzyloxy-phenyl)-1-[(1-phenyl-cyclobutymethyl)-carbamoyl}-ethyl}-methyl-carbamoyl)-2-(1-trityl-1H-imidazol-4-yl)-ethyl}-carbamicacid benzyl ester

To a suspension of Cbz-His-(Trt)-OH (synthesized according to the methodin Hudspeth J. P., Kaltenbronn J. S., Repine J. T., Roark W. H., StierM. A., Renin inhibitors III, U.S. Pat. No. 4,735,933; 1988) (1.24 g,2.33 mmol),(S)-3-(4-Benzyloxy-pheny)-2-methylamino-N-(1-phenyl-cyclobutylmethyl)-propionamidetrifluoroacetate salt (from Step 2, 2.02 mmol) and HBTU (0.97 g, 2.56mmol) in methylene chloride (20 mL), at 0° C., was added DIEA (0.86 mL,6.99 mmol). The reaction was warmed to room temperature and stirredovernight. The solution was concentrated. The residue was taken up inethyl acetate. The solution was washed with 0.5 M HCl, saturated NaHCO₃,dried over MgSO₄, filtered, and concentrated. The oil waschromatographed (CH₂Cl₂:CH₃OH 15:1) to give the desired product (1.28 g,1.36 mmol; 58% yield for Steps 2 and 3); MS-APCI 942 (m+1).

Step 4

[S-[R*,R*)]-[1-({2-(4-Benzyloxy-phenyl)-1-[(1-phenyl-cyclobutymethyl)-carbamoyl}-ethyl}-methyl-carbamoyl)-2-(3H-imidazol-4-yl)-ethyl}-carbamicacid benzyl ester

To a solution of[S-[R*,R*)]-[1-({2-(4-Benzyloxy-phenyl)-1-[(1-phenyl-cyclobutymethyl)-carbamoyl}-ethyl}-methyl-carbamoyl)-2-(1-trityl-1H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester (from Step 3, 1.28 g, 1.36 mmol) in methylene chloride(10 mL) was added TFA (10 mL). The solution was stirred at roomtemperature for 2 hours and concentrated. The residue was dissolved inethyl acetate and the solution was washed with saturated NaHCO₃, driedover MgSO₄, filtered, and concentrated. The residue was purified twiceby chromatography (first: CH₂Cl₂:CH₃OH 10:1; second: CH₂Cl₂:CH₃OH 15:1)(0.23 g, 0.33 mmol; 24% yield); MS-APCI 700 (m+1).

CHN: Calculated for C₄₂H₄₅N₅O₅.0.25H₂O C, 71.62; H, 6.51; N, 9.94.

Found: C, 71.62; H, 6.51; N, 9.91.

EXAMPLE 7[S-(R*,R*)]-(2-(3H-Imidazol-4-yl)-1-{methyl-[1-(2-methyl-2-phenyl-propyl-carbamoyl)-2-phenyl-ethyl]-carbamoyl}-ethyl)-carbamicacid benzyl ester

Step 1

Boc-NMe-Phe-NH—CH₂—C(CH₃)₂-phenyl or(S)-Methyl-[1-(2-methyl-2-phenyl-propylcarbamoyl)-2-phenyl-ethyl]-carbamicacid tert-butyl ester

Boc-NMe-Phe-OH (0.28 g, 1.0 mmol) was dissolved in methylene chloride(50 mL). N-hydroxybenzotriazole (HOBt) (0.17 g, 1.2 mmol) was addedfollowed by 0.5 M DCC/CH₂Cl₂ (2.4 mL, 1.25 mmol),β,β-dimethylphenethylamine hydrochloride (from Step 5, Example 3) (0.20g, 1.1 mmol) and DIEA (0.39 mL, 2.2 mmol). The reaction was stirred atroom temperature under nitrogen for 48 hours. The solution was filteredand concentrated in vacuo. The residue was taken up in ethyl acetate andwashed twice with 2N HCl, 1N NaHCO₃, brine, dried over MgSO₄, filtered,and concentrated. The residue was chromatographed (CHCl₃:CH₃OH 95:5)(0.32 g, 0.78 mmol, 78%). NMR confirmed the structure of the product.

Step 2

HNMe-Phe-NH—CH₂—C(CH₃)₂-phenyl trifluoroacetate or(S)-2-Methylamino-N-(2-methyl-2-phenyl-propyl)-3-phenyl-propionamide.TFA

Boc-NMe-Phe-NH—CH₂—C(CH₃)₂-phenyl (from Step 1, 0.32 g, 0.78 mmol) wastreated with 30% TFA/CH₂Cl₂ (50 mL) for 2 hours at room temperature. Thesolution was concentrated. Coevaporisation with methylene chloride wascarried out twice to give the desired product which was used withoutfurther purification. NMR confirmed the structure of the product.

Step 3

Cbz-His(Trt)-NMe-Phe-NH—CH₂—C(CH₃)₂-phenyl or[S-(R*,R*)]-[1-{Methyl-[1-(2-methyl-2-phenyl-propylcarbamoyl)-2-phenyl-ethyl]-carbamoyl}-2-(1-trityl-1H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester

HNMe-Phe-NH—CH₂—C(CH₃)₂-phenyl trifluoroacetate (from Step 2; 0.78 mmol)was dissolved in methylene chloride (40 mL). Cbz-His-(Trt)-OH(synthesized according to the method in Hudspeth J. P., Kaltenbronn J.S., Repine J. T., Roark W. H., Stier M. A., Renin inhibitors III, U.S.Pat. No. 4,735,933; 1988) (0.46 g, 0.87 mmol) was added followed by HOAt(0.23 g, 1.7 mmol), HATU (0.60 g, 1.6 mmol), and DIEA (0.95 mL, 5.5mmol). The reaction was stirred at room temperature under nitrogen for48 hours. The solution was concentrated in vacuo. The residue was takenup in ethyl acetate and washed twice with 5% citric acid, 5% NaHCO₃,brine, dried over MgSO₄, filtered, and concentrated. The product wasused without further purification. NMR confirmed the structure of theproduct.

Step 4

[S-(R*,R*)]-(2-(3H-Imadozol-4-yl)-1-{methyl-[1-(2-methyl-2-phenyl-propylcarbamoyl)-2-phenyl-ethyl]-carbamoyl}-ethyl)-carbamicacid benzyl ester

Cbz-His(Trt)-NMe-Phe-NH—CH₂—C(CH₃)₂-phenyl (from Step 3, 0.78 mmol) wastreated with 50% TFA/CH₂Cl₂ (50 mL) for 2 hours at room temperature. Thesolution was concentrated. Coevaporisation with methylene chloride wascarried out twice to give the crude product. Purification was carriedout by reverse-phase preparative HPLC (C-18 reverse-phase column, 20% to60% of 0.1%TFA in acetonitrile against 0.1%TFA in water, 100 minutes, 13mL/min) to give the desired product (0.0458 g, 10%); MS-APCI 582 (m+1).Proton NMR confirmed the structure.

CHN: Calculated for C₃₄H₃₉N₅O₄.1.59CF₃COOH.0.27H₂O C, 58.17; H, 5.40; N,9.13.

Found: C, 58.17; H, 5.40; N, 9.30.

EXAMPLE 8[S-(R*,R*)]-(2-(3H-Imidazol-4-yl)-1-{methyl-[3-methyl-1-(2-methyl-2-phenyl-propylcarbamoyl)-butyl]-carbamoyl}-ethyl)-carbamicacid benzyl ester

Step 1

Boc-NMe-Leu-NH—CH₂—C(CH₃)₂-phenyl or(S)-Methyl-[3-methyl-1-(2-methyl-2-phenyl-propylcarbamoyl)-butyl]-carbamicacid tert-butyl ester

Boc-NMe-Leu-OH (1.23 g, 5 mmol) was dissolved in methylene chloride (10mL). β,β-dimethylphenethylamine hydrochloride (Step 5, Example 3) (1.05g, 5.5 mmol) was added followed by DIEA (1.7 mL, 9.8 mmol) and HBTU (2.1g, 5.5 mmol) which was dissolved in CH₂Cl₂:DMF (1:1, 10 mL). Thereaction was stirred overnight at room temperature. The solvent wasremoved in vacuo. The residue was taken up in ethyl acetate and washedtwice with saturated NaHCO₃, brine, dried-over MgSO₄, filtered, andconcentrated (2.17 g, 5.5 mmol). The product was used without furtherpurification. NMR confirmed the structure of the product.

Step 2

HNMe-Leu-NH—CH₂—C(CH₃)₂-phenyl trifluoroacetate or(S)-4-Methyl-2-methylamino-pentanoic acid(2-methyl-2-phenyl-propyl)-amide.TFA

Boc-NMe-Leu-NH—CH₂—C(CH₃)₂-phenyl (from Step 1, 2.17 g, 5.5 mmol) wastreated with 25% TFA/CH₂Cl₂ (25 mL) for 2 hours at room temperature. Thesolvent was reduced in volume and the residue was added to 1:1hexanes:diethyl ether, which was stored overnight at −40° C. overnightto precipitate the product. The ether/hexane was decanted and theresidue dried in vacuo (1.8 g, 4.6 mmol). The product was used withoutfurther purification. NMR confirmed the structure of the product.

Step 3

Cbz-His(Trt)-NMe-Leu-NH—CH₂—C(CH₃)₂-phenyl or[S-(R*,R*)]-[1-{Methyl-[3-methyl-1-(2-methyl-2-phenyl-propylcarbamoyl)-butyl]-carbamoyl}-2-(1-trityl-1H-imidazol-4-yl)-ethyl]-carbamicacid benzyl ester

To HNMe-Leu-NH—CH₂—C(CH₃)₂-phenyl trifluoroacetate (from Step 2, 1.8 g,4.6 mmol) was added Cbz-His-(Trt)-OH (synthesized according to themethod in Hudspeth J. P., Kaltenbronn J. S., Repine J. T., Roark W. H.,Stier M. A., Renin inhibitors III, U.S. Pat. No. 4,735,933; 1988) (2.7g, 5.0 mmol) in methylene chloride (15 mL), DIEA (1.75 mL, 10 mmol), andHBTU (2.1 g, 5.5 mmol) in CH₂Cl₂:DMF (1:1, 10 mL). The reaction wasstirred overnight at room temperature. The volume of the solvent wasreduced and ethyl acetate (100 mL) and saturated NaHCO₃ (100 mL) wereadded. The organic was separated and washed twice with saturated NaHCO₃,brine, dried over MgSO₄, filtered, and concentrated to give 4.5 g ofproduct, which was used without further purification. NMR confirmed thestructure of the product.

Step 4

Cbz-His-NMe-Leu-NH—CH₂—C(CH₃)₂-phenyl or[S-(R*,R*)]-(2-(3H-Imadozol-4-yl)-1-{methyl-[3-methyl-1-2-methyl-2-phenyl-propylcarbamoyl)-butyl]-carbamoyl}-ethyl)-carbamicacid benzyl ester

Cbz-His(Trt)-NMe-Leu-NH—CH₂—C(CH₃)₂-phenyl (from Step 3, 4.5 g, 4.6mmol) was treated with 50% TFA/CH₂Cl₂. After stirring for 2 hours atroom temperature, the solvent was reduced in volume and the residue wasadded to 1:1 hexanes:diethyl ether, which was stored overnight at −40°C. to precipitate the product. The ether/hexane was decanted and theresidue dried in vacuo. Purification was carried out by reverse-phasepreparative HPLC (C-18 reverse-phase column, 20% to 60% of 0.1%TFA inacetonitrile against 0.1%TFA in water, 100 minutes, 13 mL/min) to givethe desired product (0.098 g, 0.18 mmol); MS-APCI 549 (m+1).

CHN: Calculated for C₃₁H₄₀N₅O₄.1.06CF₃COOH.0.89H₂O C, 58.21; H, 6.32; N,10.25.

Found: C, 58.21; H, 6.32;N, 10.15.

PFT Inhibitory Activity

The protein:farnesyl transferase (PFT) or farnesyl protein transferase(FPT) inhibitory activity of compounds of the present invention wereassayed in HEPES buffer (pH 7.4) containing 5 mM potassium phosphate and20 μM ZnCl₂. The solution also contained 5 mM DTT (dithiothreitol), 5 mMMgCl₂, and 0.1% PEG 8000. Assays were performed in 96-well plates(Wallec) and employed solutions composed of varying concentrations of acompound of the present invention in 100% DMSO (dimethylsulfoxide). Uponaddition of both substrates, radiolabeled farnesyl pyrophosphate ([1³H],specific activity 15 to 30 Ci/mmol, final concentration 134 nM) and(biotinyl)-Ahe-Thr-Lys-Cys-Val-Ile-Met ([3aS[3a alpha, 4 beta, 6aalpha]-hexahydro-2-oxo-1H-thieno[3,4-d]imidazole-5-pentanoicacid]-[7-aminoheptanoic acid]-Thr-Lys-Cys-Val-Ile-Met) (Ahe is7-amino-heptanoic acid, Thr is threonine, Lys is lysine, Cys iscysteine, Val is valine, Ile is isoleucine and Met is methionine) (finalconcentration 0.2 μM), the enzyme reaction was started by addition ofSF9 affinity purified rat farnesyl protein transferase. After incubationat 30° C. for 30 minutes, the reaction was terminated by diluting thereaction 2.5-fold with a stop buffer containing 1.5 M magnesium acetate,0.2 M H₃PO₄, 0.5% BSA (bovine serum albumin), and strepavidin beads(Amersham) at a concentration of 1.3 mg/mL. After allowing the plate tosettle for 30 minutes at room temperature, radioactivity was quantitatedon a microBeta counter (Model 1450, Wallec). The assay was also carriedout without 5 mM potassium phosphate.

Gel Shift Assay

Twenty-four hours after planting 2×10⁶ ras-transformed cells pertreatment condition, the farnesylation inhibitor is added at varyingconcentrations. Following an 18-hour incubation period, cells are lysedin phosphate-buffered saline containing 1% Triton X-100, 0.5% sodiumdeoxycholate, and 0.1% SDS (sodium dodecyl sulfate), pH 7.4 in thepresence of several protease inhibitors (PMSF(phenylmethylsulfonylfluoride), antipain, leupeptin, pepstatin A, andaprotinin all at 1 μg/mL). Ras protein is immunoprecipitated from thesupernatants by the addition of 3 μg v-H-ras Ab-2 (Y13-259 antibody fromOncogene Science). After overnight immunoprecipitation, 30 μL of a 50%protein G-Sepharose slurry (Pharmacia) is added followed by 45-minuteincubation. Pellets are resuspended in 2×tris-glycine loading buffer(Novex) containing 5% β-mercaptoethanol and then denatured by 5 minutesboiling prior to electrophoresis on 14% Tris-glycine SDS gels. UsingWestern transfer techniques, proteins are transferred to nitrocellulosemembranes followed by blocking in blocking buffer. Upon overnightincubation with primary antibody (pan-ras Ab-2 from Oncogene Science),an antimouse HRP (horse radish peroxidase) conjugate secondary antibody(Amersham) is employed for detection of the ras protein. Blots aredeveloped using ECL(enhanced chemiluminescence) techniques (Amersham).

Clonogenic Assay (6 Well Plates)

Sometime previous to setting up an actual test:

1. Make up 1.5% Bacto Agar in Milli-Q water and autoclave.

2. Make up 500 mL 2×DMEM-HG without phenol red by combining thefollowing:

1 bottle DMEM base powder (Sigma D-5030)

4.5 g glucose

3.7 g sodium bicarbonate

0.11 g sodium pyruvate

20 mL of 200 mM L-glutamine (Sigma G-7513)

1 mL pen-strep (GibcoBRL No. 15140-023)

Adjust pH to 7.1 with HCl; filter sterilize.

1. Set up makeshift water bath (beaker of water with thermometer, on hotplate) in the hood. Keep water temperature between 37° C. to 43° C.

2. Autoclave 1.5% Bacto Agar for approximately 2 minutes on high, oruntil completely melted. Then let it cool somewhat before using it. (Youcan keep it from resolidifying by setting the bottle on the hot plate.)

Bottom Layer (0.6% agar) Top Layer (0.3% agar) 20% calf serum 20% calfserum 40% 2X DMEM 50% 2X DMEM 40% Bacto Agar (1.5%) 20% Bacto Agar(1.5%) 10% sterile H₂O × μL cell suspension (to = 5000 cells/well)(H61cells: N1H transformed 3T3 H-ras cells)

Depending on the volume of each layer needed, use either 50 mL conicaltubes or 200 mL turnip tubes which can be floated in the “water bath”.

4. Add 1 mL of bottom layer agar/medium to each well: deliver 1 mL warmagar/medium to a well; then using the tip of the pipet, spread theagar/medium around to completely cover the bottom. Repeat with nextwell. Do not add the last mL in the pipet to a well, it leads tobubbles.

5. Allow the plates to set at room temperature for about 5 minutes untilthe bottom layer solidifies.

6. Label sterile Falcon 2054 (12×75 mm) tubes and add appropriate volumeof drug solutions into them.

7. Aliquot 4 μL of DMSO or drug solution per 1 mL of agar/medium toappropriate tubes; then add the agar/medium/cells to each tube. Alwaysadd 1 mL more than will actually be needed. Mix up and down in the pipet(gently); then deliver 1 mL to the center of each well. The upper layeris less viscous, so it will generally spread out over the bottom layerunaided. If necessary, rotate the plane of the plate gently to spreadthe top layer evenly over the bottom layer.

8. Let plates set for 5 or 10 minutes at room temperature to solidify,then put into 5% CO₂, 37° C. incubator.

9. On Day 13, add 0.5 mL of INT (tetrazolium 1 mg/mL in Milli-Q H₂O,filter sterilized) and return plates to incubator.

10. Count colonies.

The data in the table below shows farnesyl protein transferaseinhibitory activity, and activity in the gel shift and clonogenic assaysagainst ras protein of compounds of the present invention.

Example 1 was shown to have submicromolar activity as a ras farnesyltransferase inhibitor, and at a concentration of 1 μM, it was able toinhibit ras protein farnesylation (H-ras-transformed HIH 3T3 cells).Modification at the C-terminus led unexpectedly to increased activity:replacement of the 2-(phenylmethoxy)-ethylamine moiety withphenethylamine (Example 2), led to a 10-fold increase in activityagainst the enzyme and to a 5-fold increase in cellular activity. Alsothis modification led to an increase in the inhibition of colonyformation (clonogenic assay, H-ras transformed NIH 3T3 cells) from anIC₅₀ value of 15.1 μM, for Example 1, compared to an IC₅₀ value of 0.58μM, for the compound exemplified in Example 2. By further modifying theC-terminus of the compound from Example 2 (Example 3), it was possibleto unexpectedly increase the activity for the inhibition of rasfarnesylation in cells to 0.05 μM, and in the clonogenic assay an IC₅₀value of 0.10 μM was obtained. Modifications at the N-terminus andN-substituted glycine positions, were also carried out, and theseanalogues have also been shown to be unexpectedly superior as inhibitorsof ras farnesyl transferase.

IC₅₀ (μM) 5 mM PO₄•² Gel Shift (μM) Soft Agar Example Hepes Hepes M.E.D*IC₅₀ (μM) 1 7 0.4 1 15.1 2 1.4 0.032 0.2 0.58 3 0.31 0.007 0.05 0.10 40.38 0.006 ≦0.05 5 0.012 0.008 ≦0.05 6 1.01 0.013 ≦0.05 7 0.26 0.005≦0.05 8 1.76 0.010 ≦0.05 *MED is minimal effective dose to observeinhibition of ras farnesylation

2 1 6 PRT Artificial Sequence Description of Artificial Sequence 6 aminoacids on either side of organic compound 1 Thr Lys Cys Val Ile Met 1 5 26 PRT Artificial Sequence Description of Artificial Sequence 6 aminoacids on either side of organic compound 2 Thr Lys Cys Val Ile Met 1 5

What is claimed is:
 1. A compound having the Formula I

wherein: R^(a), R^(b), R^(c) are each independently C₁-C₆ alkyl orhydrogen; R^(d), R^(e), R^(f), and R^(g) are each independently C₁-C₆alkyl, hydrogen, or phenyl;

R⁴ is aryl, substituted aryl, or C₁-C₆ alkyl; and each n isindependently 0 to 5, m is 2 to 4 or a pharmaceutically acceptable saltor prodrug form thereof.
 2. The compound of claim 1 wherein Y is —O—. 3.The compound of claim 1 wherein


4. The compound of claim 1 wherein R^(a) is hydrogen, R^(b) is methyl,and R^(c) is hydrogen.
 5. The compound of claim 1 wherein


6. The compound of claim 1 wherein R³


7. The compound of claim 1 wherein


8. The compound of claim 7 wherein m of

m is 3 or
 4. 9. The compound of claim 1 wherein R³ is —(CH₂)_(n)—C₁-C₆alkyl.
 10. The compound of claim 1 wherein


11. The compound[S-(R*,R*)]-[1-[(2-(4-Benzyloxy-phenyl)-1-{[1-(2-fluoro-phenyl)-cyclopropylmethyl]-carbamoyl)}ethyl)-methyl-carbamoyl]-2-(3H-imidazol-4-yl)-ethyl]-carbamic acid benzyl ester, or a pharmaceuticallyacceptable salt form thereof.
 12. A method of inhibiting smooth muscleproliferation in a mammal which has been subjected to injury of vasculartissue comprising administering to said mammal an amount of a compoundof claim 1 for a time and under conditions effective to sufficientlyinhibit ras farnesyl transferase to inhibit said smooth muscleproliferation.